JP4878756B2 - Immobilization of enzymes used in biofuel cells and sensors - Google Patents

Description

  The present invention relates generally to fuel cells and methods of generating electricity. In particular, the present invention relates to an anode containing a biological enzyme incorporated in an immobilization material, such as an ion conducting polymer, and a method for generating electricity using it.

  Biofuel cells are similar to conventional polymer electrolyte membrane ("PEM") fuel cells in that they consist of a cathode (negative electrode) and an anode (anode) separated by a polymer electrolyte membrane. Biofuel cells differ from conventional fuel cells in the materials used to catalyze electrochemical reactions. Rather than using precious metals as catalysts, biofuel cells rely on biological molecules such as enzymes to carry out the reaction. Early biofuel cell technology used whole microbial metabolic pathways, but the problems with this method are the low capacity catalytic activity of all microorganisms and impractical power density outputs [1] . Enzymatic separation methods have spurred advances in biofuel cell applications by increasing capacity activity and catalytic capacity [1]. Separation enzyme fuel cells produce increased power density output by overcoming the obstacles of electron transfer related to cell membrane impedance and the lack of fuel-consuming microbial growth.

Enzymes are highly effective catalysts, but there have been challenges with their incorporation into fuel cells. Early enzyme-based fuel cells did not immobilize on the electrode surface, but contained the enzyme in solution [1 and the literature described therein; they are incorporated herein by reference]. The enzyme in solution is only stable for several days, but the immobilized enzyme can be stable for several months. One of the main obstacles of enzyme-based biofuel cells is to immobilize the enzyme in a membrane on the electrode surface, which extends the lifetime of the enzyme and forms a mechanically and chemically stable layer. No capacitive region is formed on the electrode surface. In most H ₂ / O ₂ fuel cells, the binder that holds the catalyst on the electrode surface is Nafion ^™ . Nafion ^™ is a perfluorinated ion exchange polymer with excellent properties as an ion conductor. However, since Nafion ^™ forms an acidic membrane that reduces the lifetime and activity of the enzyme, Nafion ^™ has not succeeded in immobilizing the enzyme on the surface of the biofuel cell electrode.

  One of the various aspects of the present invention is a biofuel cell for electricity generation comprising: a fuel fluid; an electron mediator; reducing oxidant in the presence of electrons to produce water. And (a) an electron conductor; (b) at least one enzyme capable of reacting with an oxidized form of the electron mediator and a fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator; (C) an enzyme immobilization material capable of immobilizing and stabilizing an enzyme, the enzyme immobilization material being permeable to a fuel fluid and an electron mediator, and (d) an electrocatalyst proximate to an electron conductor, The oxidized form of the electrocatalyst can react with the reduced form of the electron mediator to produce an oxidized form of the electron mediator and a reduced form of the electrocatalyst, wherein the reduced form of the electrocatalyst There bioanode having electrocatalyst that is capable of releasing electrons to the electron conductor.

  Another aspect of the present invention is a biofuel cell for electricity generation comprising: a fuel fluid; a cathode capable of reducing water to produce oxidant in the presence of electrons; and (A) an electron conductor, (b) at least one enzyme capable of reacting with an oxidized form of the electron mediator and a fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator; (c) an electron mediator; An enzyme immobilization material comprising: an enzyme immobilization material capable of immobilizing and stabilizing the enzyme and permeable to a fuel fluid; and (d) an electrocatalyst proximate to an electron conductor, The oxidized form of can react with the reduced form of the electron mediator to produce an oxidized form of the electron mediator and a reduced form of the electrocatalyst, which reduces the electrons to the electron conductor. A bioanode having an electrocatalyst that can be released.

  Another aspect of the present invention is a biofuel cell for electricity generation comprising: a fuel fluid; an electron mediator; a cathode capable of generating water by reducing an oxidant in the presence of electrons. And (a) an electron conductor, (b) at least one enzyme capable of reacting with an oxidized form of the electron mediator and a fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator, (c) An enzyme immobilization material having a micelle structure or reverse micelle structure, which is permeable to a fuel fluid and an electron mediator, and (d) an electrocatalyst proximate to an electron conductor, wherein the electrocatalyst is oxidized Can react with the reduced form of the electron mediator to produce an oxidized form of the electron mediator and a reduced form of the electrocatalyst, the reduced form of the electrocatalyst being an electron A bioanode having an electrocatalyst capable of emitting electrons to a conductor.

  Yet another aspect is a biofuel cell for electricity generation comprising: a fuel fluid; a cathode capable of reducing oxidant in the presence of electrons to produce water; and (a At least one enzyme capable of reacting with an oxidized form of the electron mediator and the fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator; and (c) an electron mediator An enzyme immobilization material comprising a micelle structure or a reverse micelle structure and permeable to a fuel fluid, and (d) an electrocatalyst proximate to an electron conductor, wherein the electrocatalyst is oxidized Can react with the reduced form of the electron mediator to produce an oxidized form of the electron mediator and a reduced form of the electrocatalyst that releases electrons to the electron conductor. A bioanode having an electrocatalyst that can be discharged.

  Another gist is a biofuel cell for electricity generation comprising: a fuel fluid; an electron mediator; a cathode capable of reducing water to produce oxidant in the presence of electrons; and A bioanode that oxidizes a fuel fluid to generate electricity, and reacts with (a) an electron conductor, (b) an oxidized form of an electron mediator and a fuel fluid, to form an oxidized form of the fuel fluid and a reduced form of the electron mediator At least one enzyme capable of producing a reduced form of an electron mediator capable of releasing electrons to an electron conductor, and (c) an enzyme immobilization material capable of immobilizing and stabilizing the enzyme comprising: A bioanode having an enzyme immobilization material that is permeable to fluid and electron mediators.

  Yet another aspect is a biofuel cell for electricity generation comprising: a fuel fluid; a cathode capable of reducing oxidant in the presence of electrons to produce water; and a fuel fluid A bioanode that oxidizes and generates electricity, reacting with (a) an electron conductor, (b) an oxidized form of an electron mediator and a fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator A reduced form of the electron mediator, at least one enzyme capable of releasing electrons to the electron conductor, and (c) an enzyme immobilization material comprising the electron mediator, which immobilizes and stabilizes the enzyme A bioanode having an enzyme immobilization material that is permeable to a fuel fluid.

  Another aspect of the present invention is a biofuel cell for electricity generation comprising: a fuel fluid; an electron mediator; a cathode capable of generating water by reducing an oxidant in the presence of electrons. And a bioanode that oxidizes the fuel fluid to generate electricity, wherein (a) the electron conductor, (b) the oxidized form of the electron mediator and the fuel fluid react with the oxidized form of the fuel fluid and the electron mediator At least one enzyme capable of producing a reduced form, wherein the reduced form of the electron mediator can release electrons to the electron conductor, and (c) an enzyme immobilization material having a micelle structure or a reverse micelle structure, A bioanode having an enzyme immobilization material that is permeable to a fuel fluid and an electron mediator.

  Another gist is a biofuel cell for electricity generation comprising: a fuel fluid; a cathode capable of reducing oxidant in the presence of electrons to produce water; and a fuel fluid. A bioanode that oxidizes to generate electricity, reacting with (a) an electron conductor, (b) an oxidized form of the electron mediator and a fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator A reduced form of the electron mediator, wherein the reduced form of the electron mediator is capable of releasing electrons to the electron conductor, and (c) an enzyme immobilization material comprising the electron mediator, having a micelle structure or a reverse micelle structure. And a bioanode having an enzyme immobilization material that is permeable to fuel fluid.

  Yet another aspect is a biofuel cell for electricity generation comprising: a fuel fluid; an electron mediator; a cathode capable of producing water by reducing an oxidant in the presence of electrons; and A bioanode that oxidizes a fuel fluid to generate electricity, wherein (a) an electron conductor, (b) an oxidized form of the electron mediator and a reduced form of the electron mediator reacting with the fuel fluid An enzyme immobilization material consisting of (c) a modified perfluorosulfonic acid-PTFE copolymer, and at least one enzyme in which the reduced form of the electron mediator can release electrons to the electron conductor, A bioanode having an enzyme immobilization material that is permeable to a fuel fluid and an electron mediator.

  Another gist is a biofuel cell for electricity generation comprising: a fuel fluid; a cathode capable of reducing oxidant in the presence of electrons to produce water; and a fuel fluid. A bioanode that oxidizes to generate electricity, reacting with (a) an electron conductor, (b) an oxidized form of the electron mediator and a fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator And an enzyme immobilization material comprising a reduced form of an electron mediator, wherein the reduced form of the electron mediator can release electrons to the electron conductor, and (c) the electron mediator, wherein the alkylammonium salt extracted perfluorosulfonic acid A bioanode having an enzyme immobilization material made of PTFE copolymer and permeable to fuel fluid.

  Another aspect of the present invention is a biofuel cell for electricity generation comprising: a fuel fluid; an electron mediator; a cathode capable of generating water by reducing an oxidant in the presence of electrons. And (a) an electron conductor, (b) at least one enzyme capable of reacting with an oxidized form of the electron mediator and a fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator; An enzyme immobilization material comprising a modified perfluorosulfonic acid-PTFE copolymer, which is permeable to a fuel fluid and an electron mediator; and (d) an electrocatalyst proximate to an electron conductor comprising: The oxidized form can react with the reduced form of the electron mediator to produce an oxidized form of the electron mediator and a reduced form of the electrocatalyst, Bioanode with electrocatalyst capable original form releasing electrons to the electron conductor.

  Yet another aspect is a biofuel cell for electricity generation comprising: a fuel fluid; a cathode capable of reducing oxidant in the presence of electrons to produce water; and (a At least one enzyme capable of reacting with the oxidized form of the electron fluid and the fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator; (c) comprising the electron mediator; An enzyme immobilization material comprising a modified perfluorosulfonic acid-PTFE copolymer and permeable to a fuel fluid; and (d) an electrocatalyst proximate to an electron conductor comprising oxidation of the electrocatalyst The form can react with the reduced form of the electron mediator to produce an oxidized form of the electron mediator and a reduced form of the electrocatalyst, the reduced form of the electrocatalyst being Bioanode with electrocatalyst capable of releasing electrons to the conductor.

  Another aspect is a method for generating electricity using any one of the biofuel cells of the above aspect of the present invention.

  Another aspect of the present invention is an enzyme immobilized in a non-natural colloidal immobilization material that can immobilize and stabilize the enzyme and is permeable to a compound smaller than the enzyme.

  Yet another gist is an enzyme immobilized in a non-cellular colloidal immobilization material that can immobilize and stabilize the enzyme and is permeable to a compound smaller than the enzyme.

  Another gist is an enzyme immobilized in a micelle or reverse micelle immobilization material that can immobilize and stabilize the enzyme and is permeable to a compound smaller than the enzyme.

  Another gist is an enzyme immobilized in a cation-modified perfluorosulfonic acid-PTFE copolymer that is able to immobilize and stabilize the enzyme and is smaller in compound permeability than the enzyme.

  Yet another aspect is a biofuel cell comprising a bioanode and a cathode, wherein (a) the bioanode is treated with carbon cloth, polymethylene green redox polymer film, salt-extracted tetrabutylammonium bromide. An alcohol dehydrogenase, and (b) the alcohol dehydrogenase is incorporated into a micelle compartment of a perfluorinated ion exchange polymer treated with salt-extracted tetrabutylammonium bromide, and (c) a polymethylene green A biofuel cell in which a redox polymer film is juxtaposed to a perfluorinated ion exchange polymer and carbon cloth treated with a salt extracted tetrabutylammonium bromide.

  Yet another aspect is a bioanode comprising a support membrane, a redox polymer, a quaternary ammonium salt modified perfluorinated ion exchange polymer and a dehydrogenase, wherein (a) the dehydrogenase is quaternary ammonium salt modified perfluorinated ion exchange. A bioanode that is incorporated into the polymer and (b) the redox polymer is juxtaposed to the support membrane and the quaternary ammonium salt modified perfluorinated ion exchange polymer.

Another gist is a bioanode comprising a carbon cloth support membrane, a polymethylene green redox polymer, a quaternary ammonium salt modified perfluorinated ion exchange polymer and an alcohol dehydrogenase, wherein (a) the dehydrogenase is modified with a quaternary ammonium salt A bioanode that is incorporated into a Nafion ^™ polymer and (b) a redox polymer is juxtaposed to a carbon fabric support membrane and a quaternary ammonium salt modified Nafion ^™ polymer.

  Yet another aspect is a perfluorinated ion exchange polymer comprising a modified and one or more enzymes, wherein the enzyme is incorporated into micelles of the modified perfluorinated ion exchange polymer. It is a polymer.

  Another aspect is a power generation method comprising oxidizing organic fuel at an anode and reducing oxygen at a cathode in the presence of a redox enzyme incorporated in the anode, wherein: a) the anode comprises an ion conducting polymer, a redox polymer membrane and a support membrane, (a) a redox enzyme is immobilized within the ion conducting polymer, and (b) a cofactor is present during the oxidation reaction at the anode. It is an electricity generation method wherein it is reduced and (c) the redox polymer membrane catalyzes the oxidation of the reduction cofactor.

Another aspect of the present invention is a power generation method comprising oxidizing alcohol at an anode and reducing oxygen at a cathode, wherein (a) the anode is a polymethylene green polymer, Comprising a quaternary ammonium bromide treated Nafion ^™ polymer, a carbon fiber support membrane and an alcohol dehydrogenase, (b) the alcohol dehydrogenase immobilized within the micellar compartment of the quaternary ammonium bromide treated Nafion ^™ polymer, and (c) the anode NOD ⁺ is reduced to NADH during the oxidation of the alcohol in (d) and (d) NADH is electrooxidized to NAD ⁺ in the polymethylene green polymer.

  One of the various aspects of the present invention is an immobilized enzyme for use in applications where increased enzyme stability is favored, particularly in biofuel electronics and biosensors. The immobilization material provides mechanical and chemical stability, thereby forming a barrier that stabilizes the enzyme over a longer period of time known so far. For purposes of the present invention, an enzyme is “stabilized” if it retains at least about 75% of its initial catalytic activity for at least about 30 days to about 365 days. Another aspect of the present invention is a fuel cell that uses an organic fuel (or a fuel fluid comprising hydrogen, ammonia or hydrocarbons) to generate electricity through an enzyme-mediated redox (oxidation / reduction) reaction. It is a fuel cell. Another aspect of the present invention is a biofuel cell comprising a bioanode and a cathode, wherein the bioanode is separated from the cathode by an electrolyte. The bioanode is fuel fluid permeable and comprises an enzyme immobilization material that immobilizes the enzyme and stabilizes the enzyme. The stability of the immobilized enzyme allows the biofuel cell to produce at least about 75% of the initial current over at least about 30 days to about 365 days.

A. Enzyme Immobilization In general, an enzyme is immobilized on an immobilization substance that immobilizes and stabilizes the enzyme. The enzyme immobilized on the immobilized substance of the present invention can be used for various applications. The enzymes and immobilization substances described below can be used for the application of the present invention.

1． Enzymes are used to catalyze the desired reaction. In general, natural enzymes, synthetic enzymes, artificial enzymes and denatured natural enzymes can be immobilized. Furthermore, engineered enzymes designed by nature or directed evolution may be used. In other words, organic or inorganic molecules that mimic enzyme properties can be used in embodiments of the present invention.

  An exemplary enzyme is oxidoreductase.

2． Enzyme Immobilization Substance The enzyme is immobilized on an enzyme immobilization substance. In one embodiment, the enzyme immobilization material is compound permeable that is smaller than the enzyme, immobilizes the enzyme, and stabilizes the enzyme. An immobilized enzyme is an enzyme that is physically restricted to a specific region of the immobilized substance while retaining its catalytic activity. There are various enzyme immobilization methods including carrier binding, cross-linking and capture. Carrier binding is the binding of an enzyme to a water-insoluble carrier. Crosslinking is intermolecular crosslinking of enzymes with bifunctional or polyfunctional reagents. Capture is the incorporation of the enzyme into the lattice of semipermeable material. For example, the enzyme can be incorporated into a semipermeable gel or confined to a reactive polymer membrane. The particular method of enzyme immobilization is not necessarily important, but the immobilization material is (1) fuel fluid permeable, (2) immobilizes the enzyme, and (3) stabilizes the enzyme.

  The immobilization material is more permeable to the compound than the enzyme. In other words, the immobilization material allows a compound smaller than the enzyme to pass through it, so that the compound can contact the enzyme immobilized on or in the immobilization material. The immobilization material can be prepared to have internal pores, channels, openings or combinations thereof, which allow movement of the compound through the immobilization material and yet within the immobilization material. Restrain the enzyme at substantially the same position.

  The immobilization material binds the enzyme to a substantially specific area of the material and allows the enzyme to retain its catalytic activity. In one embodiment, preferably the enzyme is constrained to a space of substantially the same size and shape as the enzyme, and the enzyme retains substantially all its catalytic activity. The immobilization material has pores, ducts, openings or combinations thereof, and the pores, ducts, openings and combinations thereof do not allow the enzyme to move substantially out of the space, but the enzyme Smaller compounds are allowed to contact the enzyme through the immobilized material. The anti, duct or opening has physical dimensions that meet the above requirements and depends on the size and shape of the particular enzyme to be immobilized.

  In one embodiment, it is preferred that the enzyme is placed in the pores of the immobilization material and the fuel fluid flows into and out of the immobilization material through the transport line. The relative size of the pores and the transport line can be such that the holes are large enough to immobilize the enzyme, but the transport line is too small for the enzyme to pass through it. In other embodiments, it is preferred that the transport line has a diameter of at least about 10 nm. In yet other embodiments, the pore / transport conduit diameter ratio is at least about 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1. 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, 10: 1 or more. In still other embodiments, the transport conduit has a diameter of at least about 10 nm and the pore / transport conduit diameter ratio is at least about 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1. , 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, 10: 1 or more The above is preferable.

  In addition, the immobilization material stabilizes the enzyme. The immobilization material stabilizes the enzyme by providing a chemical and mechanical barrier to protect the enzyme from denaturation. In other words, the immobilization material buffers the enzyme environment and slows enzyme denaturation. Furthermore, the immobilization material physically constrains the enzyme, and the physical constraint does not provide space for the enzyme to develop (development from a folded three-dimensional structure is a mechanism for enzyme denaturation). In one embodiment, the immobilization material preferably stabilizes the enzyme, thereby retaining its catalytic activity for at least about 30 days to about 365 days. Retention of catalytic activity is defined by the number of days that the enzyme retains at least about 75% of its catalytic activity. Enzymatic activity can be measured by chemiluminescence, electrochemistry, UV-Vis, radiochemistry or fluorescence assay, and the characteristic intensity is measured at the initial stage. Enzymes are considered to retain catalytic activity when the strength is at least 75% of the initial strength. In general, a fluorescent assay is used to measure enzyme activity. Free enzyme in solution loses its catalytic activity in hours to days. Thus, immobilization of the enzyme provides a significant benefit in stability. In other embodiments, the immobilized enzyme at least about 75% of its initial catalytic activity is at least about 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240, 270, 300, 330. For at least about 30, 45, 60, 75, 90, preferably at least about 80%, 85%, 90%, 95% or more of its initial catalytic activity. , 105, 120, 150, 180, 210, 240, 270, 300, 330, 365 days or longer.

In other embodiments, the immobilization material is a non-natural colloidal material. In yet other embodiments, the immobilization material is an acellular colloidal material such as a liposome. Non-cellular substances are not composed of cells and do not contain cells. A colloidal material is a material consisting of particles dispersed in other materials, which are too small to be resolved with a common optical microscope, but cannot pass through a semipermeable membrane. In other embodiments, the colloidal material is a material composed of particles that are substantially larger than atoms or common molecules, but are too small to be viewed with the naked eye. They may be on the order of about 10 ⁻⁷ to 10 ⁻³ centimeters and are linked or joined in various ways.

  In other embodiments, the immobilization material has a micelle or reverse micelle structure. In general, the molecules that make up micelles are amphiphilic, meaning that they contain polar hydrophilic groups and nonpolar hydrophobic groups. Molecules can aggregate to form micelles, polar groups are present on the surface of the aggregates and hydrocarbons, and nonpolar groups are sequestered in the aggregates. Reverse micelles have a reverse arrangement of polar and nonpolar groups. As long as the polar groups are close to each other and the nonpolar groups are close to each other, the amphiphilic molecules constituting the aggregate can be arranged in various ways. Furthermore, the molecules can form bilayers having non-polar groups facing each other and polar groups pointing away from each other. Alternatively, it is possible to form a bimolecular layer in which polar groups can face each other in the bimolecular layer and nonpolar groups are separated from each other.

In general, micelles or reverse micelle immobilization materials may be polymers, ceramics, liposomes, or may be formed from other molecules that form micelles or reverse micelle structures. Exemplary micelles or reverse micelle immobilization materials are: perfluorosulfonic acid-polytetrafluoroethylene (PTFE) copolymer (or perfluorinated ion exchange polymer) (Nafion ^™ or Flemion ^™ ), modified perfluoro Sulfonic acid-polytetrafluoroethylene (PTFE) copolymer (or modified perfluorinated ion exchange polymer) (modified Nafion ^™ or modified Flemion ^™ ), polysulfone, micelle polymer, block copolymer based on poly (ethylene oxide), microemulsion and / or Polymers produced from micellar polymerization and copolymers of alkyl methacrylate, alkyl acrylate and styrene. Other exemplary micelle or reverse micelle immobilization materials are: ceramic, sodium bis (2-ethylhexyl) sulfosuccinate, sodium dioctyl sulfosuccinate, lipid, phospholipid, sodium dodecyl sulfate, decyl Trimethylammonium bromide, tetradecyltrimethylammonium bromide, (4-[(2-hydroxyl-1-naphthalenyl) azo] benzenesulfonic acid monosodium salt), linoleic acid, linolenic acid, colloids, liposomes and micellar networks.

In one embodiment, preferably the micelle immobilization material is a modified perfluorosulfonic acid-PTFE copolymer (or modified perfluorinated ion exchange polymer) (modified Nafion ^™ or modified Flemion ^™ ) membrane. Perfluorinated ion exchange polymer membranes have been modified with a hydrophobic cation that is larger than ammonium (NH ₄ ⁺ ) ions. Hydrophobic cations function in several ways. First, to produce a modified perfluorosulfonic acid-PTFE copolymer (or modified perfluorinated ion exchange polymer) (Nafion ^™ or Flemion ^™ ) membrane, perfluorosulfonic acid-PTFE copolymer (or perfluorinated ion exchange). Polymer) and hydrophobic cations are mixed-casting to obtain an immobilization material, in which the pore size depends on the size of the hydrophobic cation. Therefore, the larger the hydrophobic cation, the larger the pore size. This function of the hydrophobic cation allows the pore size to be increased or decreased to suit a particular enzyme by changing the size of the hydrophobic cation.

In addition, by exchanging hydrophobic cations and protons as counter ions of the —SO ₃ ^— group on the perfluorosulfonic acid-PTFE copolymer (or perfluorinated ion exchange polymer) membrane, the hydrophobic cation becomes Alter the properties of perfluorosulfonic acid-PTFE copolymer (or perfluorinated ion exchange polymer) membranes. This change in counterion, the hydrophobic cations are considerably higher -SO ₃ than protons ^- since having affinity to a site, providing a cushioning effect in pH. This buffering action of the membrane maintains the pore pH substantially unchanged as the solution pH changes. In addition, the membrane provides a mechanical barrier that protects the enzyme immobilized on the membrane. Both buffering and mechanical barriers favor the stability of the enzyme immobilized on the modified perfluorosulfonic acid-PTFE copolymer (or perfluorinated ion exchange polymer).

The table below shows the buffering action of the modified perfluorosulfonic acid-PTFE copolymer membrane. The numbers represent the number of proton exchange sites available per gram of modified perfluorosulfonic acid-PTFE copolymer membrane, reducing the number of exchange sites available for protons and increasing the buffer capacity of the membrane for immobilized enzyme. To do. Membrane abbreviations refer to the following membranes: NH ₄ B is an ammonium bromide modified Nafion ^™ membrane; TMABr is a tetramethylammonium bromide modified Nafion ^™ membrane; TEABr is a tetraethylammonium bromide modified Nafion ^™ membrane TPropABr is a tetrapropylammonium bromide modified Nafion ^™ membrane; TBABr is a tetrabutylammonium bromide modified Nafion ^™ membrane; TpentABr is a tetrapentylammonium bromide modified Nafion ^™ membrane.

In order to prepare a modified perfluorosulfonic acid-PTFE copolymer (or perfluorinated ion exchange polymer) membrane, in the first step, perfluorosulfonic acid-PTFE copolymer (or perfluorinated ion exchange polymer), especially Nafion ^™ A suspension and a solution of hydrophobic cation are cast to form a membrane. After excess hydrophobic cations and their salts are extracted from the initial membrane, the membrane is recast. When recast, the membrane contains a hydrophobic cation associated with the —SO ₃ ^— site of the perfluorosulfonic acid-PTFE copolymer (or perfluorinated ion exchange polymer) membrane.

To produce a more stable and reproducible quaternary ammonium salt treated Nafion ^™ membrane, excess bromide salt should be removed from the casting solution. This salt extraction membrane is formed by re-casting the mixed cast membrane after extracting excess quaternary ammonium bromide and HBr salts from the initial membrane. Membrane salt extraction maintains the presence of quaternary ammonium cations at the sulfonic acid exchange sites, but over-completions may be trapped in the pores or create voids in the equilibrium membrane. Remove from. The chemical and physical properties of salt extraction membranes have been characterized by voltammetry, ion exchange force measurements and fluorescence microscopy prior to enzyme immobilization [3].

Illustrative hydrophobic cations are: ammonium-based cation, quaternary ammonium cation, alkyltrimethylammonium cation, alkyltriethylammonium cation, organic cation, phosphonium cation, triphenylphosphonium, Pyridinium cation, imidazolium cation, hexadecylpyridinium, ethidium, viologen, methyl viologen, benzyl viologen, bis (triphenylphosphine) iminium, metal complex, bipyridyl metal complex, metal complex based on phenanthroline, [Ru (bipyridine) ₃ ] ²⁺ and [Fe (phenanthroline) ₃ ] ³⁺ .

In one embodiment, it is preferred that the large hydrophobic cation is an ammonium-based cation. In particular, large hydrophobic cations are quaternary ammonium cations. In other embodiments, the quaternary ammonium cation is represented by Formula 1 below:

Wherein R ₁ , R ₂ , R ₃ and R ₄ are independently hydrogen, hydrocarbyl, substituted hydrocarbyl or heterocyclo, and at least one of R ₁ , R ₂ , R ₃ and R ₄ is other than hydrogen Is. In other embodiments, preferably R ₁ , R ₂ , R ₃ and R ₄ are independently hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl, and R ₁ , R ₂ , R ₃ and R ₄ are other than hydrogen. In yet other embodiments, R ₁ , R ₂ , R ₃ and R ₄ are the same and are methyl, ethyl, propyl, butyl, pentyl or hexyl. In yet other embodiments, preferably R ₁ , R ₂ , R ₃ and R ₄ are butyl.

FIGS. 4-6 show fluorescence micrographs of enzymes immobilized on various modified perfluorosulfonic acid-PTFE copolymer membranes treated with a solution of NAD ⁺ and fuel fluid in pH 7.15 phosphate buffer. Fluorescence micrographs showing fluorescence show that the enzyme immobilized on the specific modified perfluorosulfonic acid-PTFE copolymer membrane is still catalytically active after immobilization. This is one way of determining whether a particular immobilization material will immobilize and stabilize an enzyme while maintaining the catalytic activity of the enzyme.

Mixed cast films of quaternary ammonium salts (eg, tetrabutylammonium bromide) and Nafion ^™ increase the mass transport of small analytes through the film and reduce the selectivity of the membrane for anions [2]. These membranes have conductivities very similar to unmodified Nafion, but rather than protons to quaternary ammonium bromide than protons, as shown by titrating the number of usable exchange sites for protons in the membrane. It has a very high selectivity. Thus, these films have similar electrical properties but very different acid / base properties. The treated membrane maintains a neutral pH over a wide range of buffer pH.

B. Biofuel cell One of the various gist of the present invention is a biofuel cell using the above-mentioned immobilized enzyme. In biofuel cells, the reaction that occurs at the anode is the oxidation of the fuel fluid with the simultaneous emission of electrons; the electrons are directed to the cathode by electrical coupling, where the oxidant is reduced to water. The biofuel cell of the present invention provides an energy source (electricity) to an electrical load external to the biofuel cell. To facilitate the oxidation of the fuel fluid, the bioanode contains an electron conductor, an electrocatalyst for an electron mediator (also known as a cofactor) (eg, a redox polymer), and an enzyme. An electron mediator is a compound that can accept or donate electrons.

  First, the oxidized form of the electron mediator is reacted with the fuel fluid and the enzyme to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator. Next or simultaneously, the reduced form of the electron mediator is reacted with an oxidized form of the electrocatalyst to produce an oxidized form of the electron mediator and a reduced form of the electrocatalyst. Next, the reduced form of the electrocatalyst is oxidized at the anode to generate electrons and generate electricity. The redox reaction at the bioanode can be reversible except for the oxidation of the fuel fluid, so that no enzymes, electron mediators and electrocatalysts are consumed. When an electron mediator is added to give an additional reactant, the redox reaction can optionally be made irreversible.

  Alternatively, electron conductors and enzymes can be used, in which case, at a non-modified carbon-based electrode, an electron mediator that contacts the bioanode can transfer electrons between its oxidized and reduced forms. In an unmodified carbon-based electrode, if the electron mediator can transfer electrons between its oxidized and reduced forms, the reaction of the next electrocatalyst with the electron mediator is not necessary, and the electron mediator itself is at the anode. Oxidizes to generate electrons, or electricity.

  The biofuel cell of the present invention has a bioanode and a cathode. Electron emission occurs at the bioanode, oxidant reduction occurs at the cathode, and an electrical load is placed between the bioanode and the cathode.

1． Bioanode In general, a bioanode has a component that oxidizes a fuel fluid where electrons are emitted and directed to an external electrical load. An electrical load is a device that uses electrons. An external electrical load is in contact with the cathode where the oxidant is reduced. This flow of electrons from the fuel fluid to the bioanode, electrical load and cathode by electrical coupling provides an energy source (electricity) to the electrical load.

  In one embodiment, the bioanode comprises an electron conductor and an enzyme immobilized on an enzyme immobilization material. In other embodiments, the bioanode optionally further comprises an electrocatalyst for the electron mediator. If the bioanode is in contact with an electron mediator capable of undergoing a reversible redox reaction in the electron conductor, the electrocatalyst may not be present in the bioanode. The components of the bioanode are in close proximity to each other, meaning that they are physically or chemically bound by appropriate means. In one embodiment, the components are physically and chemically coupled by placing them in solution by electrical coupling between them. In other embodiments, it is preferred to physically bond the components by coating or otherwise attaching the individual components to the electronic conductor. The components can be deposited separately, for example in layers, or incorporated into one deposition layer.

a. Electron Conductor An electron conductor is a substance that conducts electrons. The electron conductor may be inorganic or organic in nature as long as it can conduct electrons into the material. The electron conductor may be a carbonaceous material, a metallic conductor, a semiconductor, a metal oxide or a modified conductor.

Particularly suitable electron conductors are carbon-based materials. Exemplary carbon-based materials are: carbon cloth, carbon paper, carbon screen printed electrode, carbon paper (Toray), carbon paper (ELAT), carbon black (Vulcan XC-72, E-tek), Carbon black, carbon powder, carbon fiber, single-walled carbon nanotube, double-walled carbon nanotube, multi-walled carbon nanotube, carbon nanotube array, diamond-coated conductor, glassy carbon and mesoporous carbon. In addition, other exemplary carbonaceous materials include graphite, uncompressed graphite worms, delaminated flake graphite (Superior ^TM graphite), high performance graphite and carbon powder (Formula BT ^TM , Superior ^TM graphite), highly ordered pyrolysis Graphite, pyrolytic graphite and polycrystalline graphite.

  In still other embodiments, the electronic conductor can be manufactured from a metallic conductor, and suitable electronic conductors include gold, platinum, iron, nickel, copper, silver, stainless steel, mercury, tungsten, and others suitable for electrode configurations. Can be made from any metal. Furthermore, electronic conductors that are metallic conductors can be composed of nanoparticles made from cobalt, diamond, and other suitable metals. The other metallic electronic conductor may be a silver plated nickel screen printed electrode.

  Furthermore, the electronic conductor may be a semiconductor. Suitable semiconductors are made from silicon and germanium and can be doped with other components. The semiconductor can be doped with phosphorus, boron, gallium, arsenic, indium or antimony, or combinations thereof.

  Other electron conductors may be metal oxides, metal sulfides, main group element compounds, and materials modified with electron conductors. Exemplary electronic conductors of this type are: nanoporous titanium oxide, tin oxide coated glass, cerium oxide particles, molybdenum sulfide, boron nitride nanotubes, aerogels modified with conductive materials such as carbon, such as carbon Sol gel modified with a conductive material, ruthenium carbon aerogel, and mesoporous silica modified with a conductive material such as carbon.

  In other embodiments, the electronic conductor has two or more components, the first component may be a carbon cloth, and the second component may be the electronic conductor described above. A second electronic conductor is attached to the carbon cloth, and the two combinations constitute an electronic conductor.

In other embodiments, the electronic conductor is composed of an uncompressed (GAT ^™ expanded, Superior Graphite Co.) graphite worm, as described in Section B.1.c below.

b. Electron Mediator An electron mediator is a compound that can accept or donate electrons. In other words, the electron mediator has an oxidized form that can accept electrons to form a reduced form, and the reduced form can donate electrons to form an oxidized form. An electron mediator is a compound that can diffuse into and / or be incorporated into an immobilization material.

  In one embodiment, the diffusion coefficient of the electron mediator is maximized. In other words, the mass transfer of the reduced form of the electron mediator is made as fast as possible. The fast mass transfer of the electron mediator allows for greater circuit potential difference and power density of the biofuel cell in which it is used.

Exemplary electron mediators are nicotinamide adenine dinucleotide (NAD ⁺ ), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADP) or pyrroloquinoline quinone (PQQ) or their equivalents. Other exemplary electron mediators are phenazine methosulfate, dichlorophenolindophenol, short chain ubiquinone and potassium ferricyanide.

  In one embodiment, the electron mediator cannot undergo a redox reaction alone in the electron conductor. In this embodiment, the bioanode comprises an electrocatalyst for an electron mediator that promotes the emission of electrons in the electron conductor.

  In another embodiment, a reversible redox couple having a standard reduction potential difference of 0.0V ± 0.5V is used as the electron mediator. Therefore, an electron mediator that gives reversible electrochemistry to the surface of the electron conductor can be used. The electron mediator binds to a natural enzyme dependent on the electron mediator, an enzyme modified depending on the electron mediator, or a synthetic enzyme dependent on the electron mediator. Examples of electron mediators that impart reversible electrochemistry to the electron conductor surface are pyrroloquinoline quinone (PQQ), phenazine methosulfate, dichlorophenol indophenol, short chain ubiquinone and potassium ferricyanide. In one embodiment, it is preferred that the electron mediator used with the bioanode is PQQ. The ability of the electron mediator to provide reversible electrochemistry on the surface of the electron conductor eliminates the need for an electrocatalyst that catalyzes the redox reaction of this type of electron mediator.

c. Electrocatalysts for electron mediators Generally, electrocatalysts are substances that promote the emission of electrons in an electron conductor. In other words, the electrocatalyst increases the reaction rate of the reduction or oxidation of the electron mediator, whereby the reduction or oxidation of the electron mediator can occur at a lower reduction potential difference. The electrocatalyst can be reversibly oxidized at the anode to generate electrons, ie electricity. When the electrocatalyst is in close proximity to the electronic conductor, the electrocatalyst and the electronic conductor are in electrical contact with each other, but not necessarily in physical contact with each other. In one embodiment, the electron conductor is associated with or in close proximity to an electrocatalyst for the electron mediator.

In general, the electrocatalyst may be an azine, a conductive polymer or an electroactive polymer. Examples of electrocatalysts are: methylene green, methylene blue, luminol, nitro-fluorenone derivatives, azine, osmium phenanthrolinedione, catechol-pendant terpyridine, toluene blue, cresyl blue, nile blue, neutral red, phenazine derivatives, thionine , Azure A, Azure B, Toluidine Blue O, Acetophenone, Metallophthalocyanine, Nile Blue A, Modified transition metal ligand, 1,10-phenanthroline-5,6-dione, 1,10-phenanthroline-5,6-diol, [ Re (phen-dione) (CO) ₃ Cl], [Re (phen-dione) ₃ ] (PF ₆ ) ₂ , poly (metallophthalocyanine), poly (thionine), quinone, diimine, diaminobenzene, diaminopyridine, phenothiazine , Fenoki Sajin, toluidine blue, brilliant cresyl blue, 3,4-dihydroxybenzaldehyde, poly (acrylic acid), poly (azul I), poly (nile blue A), poly (methylene green), poly (methylene blue), polyaniline, polypyridine , Polypyrrole, polythiophene, poly (thieno [3,4-b] thiophene), poly (3-hexylthiophene), poly (3,4-ethylenedioxypyrrole), poly (isothianaphthene), poly (3,4 -Ethylenedioxythiophene), poly (difluoroacetylene), poly (4-dicyanomethylene-4H-cyclopenta [2,1-b; 3,4-b '] dithiophene), poly (3- (4-fluorophenyl) Thiophene) and poly (neutral red).

  In another embodiment, preferably the electronic conductor consists of an uncompressed graphite worm, which is treated with an electrocatalyst (or redox polymer) for an electronic mediator, in particular methylene green, before assembly, such as a hydraulic press. It is pressed into the desired shape by conventional means. The electronic conductor pressing conditions were optimized to produce a soft material of sufficient thickness. After compression of the electron conductor, methylene green was polymerized by cyclic voltammetry in a solution of methylene green. After the electronic conductor was washed and dried, the final material was adhered to the carbon cloth.

d. Enzymes are required to catalyze the oxidation of fuel fluids. In general, the aforementioned enzymes can be used. An exemplary enzyme used in the bioanode is oxidoreductase. In one embodiment, it is preferred that the oxidoreductase acts on the CH—OH or CH—NH group of the donor.

  In other embodiments, it is preferred that the enzyme is a dehydrogenase. In other embodiments, the enzyme is: alcohol dehydrogenase, aldehyde dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, glucose dehydrogenase, glucose oxidase, lactate dehydrogenase, lactose dehydrogenase or pyruvate dehydrogenase. In yet another embodiment, it is more preferred that the enzyme is alcohol dehydrogenase. In yet other embodiments, the enzyme is a PQQ-dependent dehydrogenase.

e. Enzyme immobilization material Enzyme immobilization material is used in biofuel cells. In one embodiment, the enzyme immobilization material is fuel fluid permeable and immobilizes and stabilizes the enzyme. The enzyme immobilization materials can be used in biofuel cell embodiments when they are fuel fluid permeable. The immobilization material is permeable to the fuel fluid, so that the oxidation reaction of the fuel can be catalyzed by the immobilized enzyme. In other words, the immobilization material allows movement of the fuel fluid therethrough, thereby allowing the fuel to contact the enzyme immobilized on or within the immobilization material. The immobilization material can be prepared to have internal pores, conduits, openings or combinations thereof that allow for movement of the fuel fluid through the immobilization material and substantially within the immobilization material. Restrain the enzyme in the same space.

  In other embodiments, it is preferred that the enzyme be placed in the pores of the immobilization material so that the fuel fluid flows in and out of the immobilization material through the transport line. The relative size of the pores and the transport line can be such that the holes are large enough to immobilize the enzyme, but the transport line is too small for the enzyme to pass through it. In other embodiments, it is preferred that the transport line has a diameter of at least about 10 nm. In yet other embodiments, the pore / transport conduit diameter ratio is at least about 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5: 1. 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, 10: 1 or more. In still other embodiments, the transport conduit has a diameter of at least about 10 nm and the pore / transport conduit diameter ratio is at least about 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4: 1. , 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5: 1, 10: 1 or more The above is preferable.

f. Bioanode Embodiment In another embodiment, it is preferred that the bioanode comprises an electron conductor modified by adsorbing, polymerizing or covalently bonding an electrocatalyst for an electron mediator to the electron conductor. This embodiment has the advantage of increasing the surface area of the electronic conductor. Prior to construction, treatment of the electron conductor by adsorbing the electrocatalyst on the surface of the electron conductor and then chemically or electrochemically polymerizing the electrocatalyst has a higher catalytic activity compared to the untreated electron conductor. give.

  In other embodiments, it is more preferred that the bioanode consist of an electronic conductor consisting of an uncompressed graphite worm that has been treated with methylene green prior to construction and pressed into the desired shape with a hydraulic press. After pressing the electron conductor, methylene green was polymerized by cyclic voltammetry in a solution of methylene green. After the electronic conductor was washed and dried, the final material was adhered to the carbon cloth to complete the electronic conductor. Next, in order to produce a TBAB-modified perfluorosulfonic acid-PTFE copolymer (or TBAB-modified perfluorinated ion exchange polymer) membrane, a perfluorosulfonic acid-PTFE copolymer (or perfluorosulfonic acid-PTFE copolymer) modified with tetrabutylammonium bromide (TBAB) is used. An ionized ion exchange polymer) membrane was prepared as described above and in the Examples. After recasting, the enzyme was added to the solution. This TBAB-modified perfluorosulfonic acid-PTFE copolymer (or TBAB-modified perfluorinated ion exchange polymer) membrane containing immobilized enzyme was pipetted onto and / or painted and dried on an electronic conductor.

  In one embodiment, the bioanode and cathode are preferably configured in a membrane electrode assembly (MEA). In this embodiment, the cathode may be a common cathode, such as a platinum doped (single or double doped) gas diffusion electrode, or a biocathode as described below. In general, the cathode was placed below, a sheet of perfluorosulfonic acid-PTFE copolymer was placed on the cathode, and the bioanode was placed on the membrane. The whole was placed between the heating elements of the hydraulic press; pressure was applied thereby causing the top and bottom plates of the press to come into contact, then the press was heated and finally more pressure was applied. Alternatively, the MEA can be assembled in the same manner as described above except between: the bioanode and one perfluorosulfonic acid-PTFE copolymer membrane, and the cathode and one perfluorosulfonic acid-PTFE copolymer membrane. A few drops of liquid perfluorosulfonic acid-PTFE copolymer membrane or modified perfluorosulfonic acid-PTFE copolymer membrane was applied and pressed as described above without heating.

  In other embodiments, the electron mediator can be physically linked to the enzyme. The physical bond may be a covalent bond or an ionic bond between the electron mediator and the enzyme. In yet another aspect, when the electron mediator is reversible electrochemical at the electron conductor, the electron mediator can be physically bound to the enzyme, and the electron mediator can be physically bound to the electron conductor.

In yet another embodiment, the electron mediator is immobilized on an immobilization material. In yet another embodiment, oxidized NAD ⁺ is preferably immobilized on a cation-modified perfluorosulfonic acid-PTFE copolymer (cation-modified Nafion ^™ ) membrane. After the fuel fluid is added to the cell, NAD ⁺ is reduced to NADH, which can diffuse into the cation-modified perfluorosulfonic acid-PTFE copolymer (cation-modified Nafion ^™ ) membrane.

In other embodiments, the present invention provides for immobilizing a dehydrogenase enzyme to a salt extracted tetrabutylammonium / perfluorinated ion exchange polymer membrane (eg, Nafion ^™ membrane or Flemion ^™ membrane [Asahi Glass Co., Tokyo]). Including. The salt extraction polymer suspension is neutral and a buffered enzyme solution can be added to this suspension. The mixture can be cast onto the anode to form a modified electrode, immobilizing the enzyme near the electrode surface.

In another embodiment, the present invention relates to a perfluorinated ion comprising a modification that produces a neutral pH in the micelle of the polymer and one or more enzymes incorporated into the micelle of the modified perfluorinated ion exchange polymer. Relates to exchange polymers. A preferred perfluorinated ion exchange polymer is a Nafion ^™ polymer. Preferred enzymes are redox enzymes such as dehydrogenases that catalyze the oxidation of organic fuels and cofactor reduction. Cofactors include NAD ⁺ , NADP ⁺ and FAD.

In one embodiment, the present invention relates to a fuel cell comprising a bioanode and a cathode, wherein the bioanode comprises a redox polymer film, a modified ion exchange polymer membrane and a dehydrogenase. Dehydrogenase is incorporated into the micellar compartment of the modified ion exchange polymer membrane. Preferably, the modified ion exchange polymer membrane is a perfluorinated ion exchange polymer treated with a salt extracted quaternary ammonium. Commercially available perfluorinated ion exchange polymers include Nafion ^™ (DuPont) and Flemion ^™ (Asahi Glass Co., Ltd.). Preferably, the perfluorinated ion exchange polymer is a Nafion ^™ polymer or a Flemion ^™ polymer. Preferred quaternary ammonium salts include tetrabutylammonium bromide. A preferred redox polymer film is polymethylene green. The bioanode may comprise two or more different dehydrogenases, such as alcohol dehydrogenase and aldehyde dehydrogenase.

2． Fuel fluid Fuel fluid is consumed in the oxidation reaction between the electron mediator and the immobilized enzyme. The fuel fluid size is small enough to increase the diffusion coefficient into the immobilized material. Illustrative fuel fluids are: hydrogen, ammonia, alcohol (eg, methanol, ethanol, propanol, isobutanol, butanol and isopropanol), allyl alcohol, aryl alcohol, glycerol, propanediol, mannitol, glucuronate, aldehyde, Carbohydrates (e.g. glucose, glucose-1, D-glucose, L-glucose, glucose-6-phosphate, lactate, lactate-6-phosphate, D-lactate, L-lactate, fructose, galactose-1, galactose, aldose, Sorbose and mannose), glycerate, coenzyme A, acetyl Co-A, malate, isocitrate, formaldehyde, acetaldehyde, acetate, citrate, L-glu Conate, β-hydroxy steroid, α-hydroxy steroid, lactaldehyde, testosterone, gluconate, fatty acid, lipid, phosphoglycerate, retinal, estradiol, cyclopentanol, hexadecanol, long chain alcohol, coniferyl-alcohol, cinnamyl-alcohol , formate, long chain aldehydes, pyruvate, butanal, acyl-CoA, steroids, amino acids, _{flavin, NADH, NADH 2, NADPH,} NADPH 2 and hydrogen.

  In other embodiments, preferably the fuel fluid is an alcohol, more preferably methanol and ethanol, especially ethanol.

3． The cathode cathode is a common cathode or biocathode. In a typical cathode, the cathode is based on platinum and is exposed to air to reduce the oxidant. If a common cathode is used, it is separated from the anode by a salt bridge. Salt bridges can take a variety of forms and are materials that allow ions to move between the anode and the cathode. In one embodiment, it is preferred that the salt bridge is a polymer electrolyte membrane (PEM).

In one embodiment, the fuel electrons may comprise the bioanode and biocathode described above, wherein the biocathode comprises a second redox polymer film, a second modified ion exchange polymer membrane, and O ₂ -reductase. . The second modified ion exchange polymer membrane may be a salt extracted quaternary ammonium treated Nafion ^™ polymer, the second redox polymer film may be poly (N-vinyl-imidazole) and the O ₂ -reductase is laccase. It's okay. According to this embodiment, where the biofuel cell comprises both a bioanode and a biocathode, no “salt bridge” or PEM is required to separate the anode from the cathode. This embodiment is particularly useful as an implantable power source for biological hosts such as humans.

Four. One of the various aspect of the electricity generation process of the present invention is a method of generating power, the bioanode of the present invention to oxidize organic fuel (or hydrogen, the fuel fluid comprising ammonia or hydrocarbons), A method of using an oxidant such as oxygen or peroxide with a reducing cathode. Furthermore, another aspect of the present invention is a method for generating electricity using the bioanode and cathode, wherein an electrical load is disposed between the bioanode and the cathode, and the bioanode, electrical load and cathode are In this method, the conductive materials are connected. The fuel fluid is oxidized at the bioanode and the oxidant is reduced at the cathode. An electrical load is a device that uses electricity. In one embodiment, the bioanode is in contact with a solution containing a fuel fluid and an electron mediator, and the cathode is in contact with a buffer solution. The solution in contact with the bioanode is separated from the solution in contact with the cathode by salt bridge or PEM.

  In another aspect, a power generation method comprises oxidizing a fuel fluid at a bioanode and reducing an oxidant at a cathode, wherein (a) the bioanode is for an electron conductor, an electron mediator. An electrocatalyst, and an enzyme immobilized in an enzyme immobilization material that is permeable to the fuel fluid and stabilizes the enzyme, and (b) an oxidized form of the electron mediator is present in the oxidation of the fuel fluid at the bioanode. And (c) the reduced form of the electron mediator is oxidized by the electrocatalyst, and then or simultaneously, the electrocatalyst is oxidized at the bioanode.

  In yet another aspect, the power generation method comprises the steps of oxidizing the fuel fluid at the bioanode and reducing the oxidant at the cathode, wherein (a) the bioanode comprises an electronic conductor and a fuel fluid. An electron mediator that is permeable and comprises an enzyme immobilized in an enzyme immobilization material that stabilizes the enzyme, which can be reversible electrochemistry in the electron conductor, is present in the solution in contact with the bioanode, ( b) The oxidized form of the electron mediator is reduced during the oxidation of the fuel fluid at the bioanode, and (c) the electron mediator is oxidized at the bioanode.

  In yet another aspect, the method for generating power comprises oxidizing the fuel fluid at the bioanode and reducing the oxidant at the cathode, wherein (a) the bioanode is for an electron conductor, an electron mediator. And (b) an oxidized form of the electron mediator is reduced during oxidation of the fuel fluid at the bioanode, and (b) an enzyme immobilized on a micelle or reverse micelle enzyme immobilization material, c) The reduced form of the electron mediator is oxidized by the electrocatalyst, which is then oxidized at the bioanode.

  In yet another embodiment, the method for generating power comprises oxidizing the fuel fluid at the bioanode and reducing the oxidant at the cathode, wherein (a) the bioanode comprises an electron conductor, and a micelle or An electron mediator comprising an enzyme immobilized on a reverse micelle enzyme immobilization material, which can be reversible electrochemistry in the electron conductor, is present in the solution in contact with the bioanode, and (b) the oxidized form of the electron mediator is Reduced during oxidation of the fuel fluid at the bioanode, and (c) the electron mediator is oxidized at the bioanode.

In yet another embodiment, the present invention is a method of generating power comprising oxidizing organic fuel at an anode in the presence of a redox enzyme incorporated into the anode; and reducing oxygen at the cathode; Relates to a method comprising: A preferred power generation method comprises the steps of oxidizing alcohol at the anode and reducing oxygen at the cathode, wherein (a) the anode is a polymethylene green polymer, a quaternary ammonium bromide treated Nafion ^™ polymer. Comprising a carbon fiber support membrane and an alcohol dehydrogenase; (b) the alcohol dehydrogenase is immobilized in the micellar compartment of a quaternary ammonium bromide treated Nafion ^™ polymer, and (c) NAD ⁺ is oxidized of alcohol at the anode (D) NADH is electrooxidized to NAD ⁺ in the polymethylene green polymer.

In accordance with the present invention, a biofuel cell was constructed using a dehydrogenase enzyme-immobilized ion conducting membrane cast on the surface of a poly (methylene green) modified anode. In one embodiment of the invention, the alcohol fuel is oxidized to an aldehyde in the presence of alcohol dehydrogenase and NAD ⁺ . The NADH product is a redox mediator for fuel cells. Since NADH oxidation at platinum and carbon electrodes has a low reaction rate and occurs at large overvoltages [4], polymer-based electrocatalysts were used to regenerate NAD ⁺ and transfer electrons from NADH to the electrode. . The electrocatalyst is preferably poly (methylene green) [5].

Ethanol / O ₂ biofuel cells using these bioanodes incorporating aldehyde dehydrogenase in addition to alcohol dehydrogenase have power densities of 1.16 mW / cm ² depending on the alcohol dehydrogenase / aldehyde dehydrogenase ratio in the polymer layer. 2.04 mW / cm ² and an open circuit voltage of 0.6 V to 0.82 V are generated. Methanol / O ₂ biofuel cells using these bioanodes produce a power density of 1.55 mW / cm ² and an open circuit voltage of 0.71V.

  In one embodiment of the present invention, incorporating a redox enzyme into a modified ion conducting polymer having a near neutral pH allows for increased power density of the biofuel cell and increased life of the bioanode.

C. In other embodiments of the enzyme sensor , the immobilized enzyme of the present invention can be used as a sensor. An enzyme sensor is a sensor in which a chemical species (analyte) to be measured undergoes an enzyme-catalyzed reaction at the sensor before detection. Reaction of the analyte with the enzyme (the analyte should be a substrate for the enzyme) yields a secondary species, and the concentration of the secondary species is proportional to or the same as the concentration of the analyte. . The concentration of secondary species is then detected electrochemically or spectroscopically by means of a transducer, for example by means of electrodes or optical fibers.

  The enzyme of the enzyme sensor is generally contained in a membrane suitable for contacting the test fluid. In this case, the enzyme can be included as part of the sensor membrane or incorporated into the sensor proper, eg as part of the electrode layer. Thus, after diffusion to the outside of the sensor, the analyte contacts the enzyme, an enzyme / analyte reaction occurs, and then secondary species diffuse to the detector portion of the sensor. The immobilized enzyme in the micelle or reverse micelle immobilization material has a suitable porosity, so that the analyte can controllably diffuse from the test fluid to the enzyme and at the same time retain the enzyme Thus preventing the enzyme from being leached into the test fluid.

  In one embodiment, a glassy carbon electrode was modified with methylene green and polymerized as described above. In addition, a glassy carbon electrode was coated with a tetrabutylammonium bromide-modified perfluorosulfonic acid-PTFE copolymer film on which alcohol dehydrogenase was immobilized. The enzyme sensor detected ethanol.

D. Use of immobilized enzyme In general, the immobilized enzyme of the present invention can be used in various applications. Applications where it is advantageous to immobilize or stabilize the enzyme are of particular interest.

  In other embodiments, immobilized enzymes are used in bioreactors and bioprocesses. An enzyme immobilized on an immobilization substance such as a micelle or reverse micelle immobilization substance acts to induce a chemical reaction of a substrate by catalysis. The substrate of the present invention is any substrate on which the immobilized enzyme can catalyze a chemical reaction. Thus, the feed stream containing the substrate contacts the immobilized enzyme, where the substrate reacts to form a product. Furthermore, the substrate physically separates from the product as it passes through the immobilization material, where unreacted substrate is retained and passes through the exit end of the separation and collection housing.

  The method using a bioreactor can be arranged as a one-pass, continuous feed system or recirculation system, where unreacted substrate is returned to the feed stream and the product passes through the immobilized enzyme. Collected separately. The retained and permeable species are generally physically separated by size, with smaller products passing through the immobilized material and larger substrates being prevented from passing through the immobilized material.

  In yet another embodiment, the immobilized enzyme of the invention is used in a bioassay. Bioassay procedures include the reaction of a test substance with an appropriate reagent, such as an enzyme substrate, or vice versa, as well as the characteristics of the reagent (or other substances that react with the reagent, indicators) such as color, radioactivity or Includes direct or indirect quantitative measurement of the amount of reacted reagent by measurement of other physical characteristics. The test solution is contacted with the immobilized enzyme, which causes a reaction to produce an immobilized complex. Radiolabeled substrate is introduced and allowed to react at unoccupied sites. By measuring the radioactivity in the complex, the amount of enzyme in the sample can be measured.

  In yet other embodiments, immobilized enzymes are used for enzyme therapy. Enzymatic therapy consists of the administration of an enzyme to a patient, which catalyzes an internal reaction that the patient's own defective or deleted enzyme cannot catalyze. Immobilized enzymes are advantageous for this application because of the increased stability and allow the administered enzyme to remain active longer, thus reducing the frequency of treatment and reducing enzyme dosage. it can.

  In other embodiments, the immobilized enzyme of the invention is used in an immunoassay. One such assay is a quantitative enzyme-linked immunosorbent assay (ELISA), in which color can be detected visually on filter paper, porous membranes, plastic paddles or other solid surfaces. . This assay can be readily adapted for quantitative assays and the use of other detectable signals in addition to color.

  In other embodiments, the immobilized enzyme is used as a biomimetic. In a biomimetic, a synthetic system that mimics a biological or natural system is prepared. For example, to mimic a cell line or a larger biological system, such as a cardiovascular or endocrine system, the biomimetic system needs to be able to selectively react with a specific substance and release the substance from the biomimetic system. is there. One way to selectively react and release the substance is to use an immobilized enzyme immobilized on a semipermeable membrane. In this method, for example, a semi-permeable membrane mimics a cell membrane, and an enzyme-mediated reaction mimics many enzyme-mediated reactions that occur in natural systems.

  In other embodiments, the immobilized enzyme is used in a system that mimics the Krebs cycle (or citrate cycle). The Krebs cycle involves the oxidation of acetic acid or acetyl-CoA to carbon dioxide and water. The Krebs cycle biomimetic provides information about factors that affect the energy production cycle of cells. In addition, research on the Krebs cycle and its biomimetics will help understand aspects that are important for maximizing energy production using biomimetics.

Definitions As used herein, a “fuel cell” has an anode and a cathode that are separated to prevent electrical shorting. Preferably, the anode and cathode are separated by a polymer electrolyte membrane. Biofuel cells use fuel fluids and enzymes that catalyze the oxidation of fuel fluids. In one embodiment, a “biofuel cell” uses an organic fuel as an energy source and a redox enzyme that catalyzes the oxidation of the organic fuel. The terms “fuel cell” and “biofuel cell” are used interchangeably in the present disclosure. In one embodiment, the fuel cell of the present invention may be used in applications requiring an electrical supply, such as, but not limited to, appliances, toys, internal medical devices, and electric (electric) vehicles. In another aspect, the fuel cell of the present invention can be implanted in a living body, in which case the organic fuel is derived from the living body and the fuel cell supplies power to the device implanted in the living body.

  As used herein, the term “organic fuel” means any carbon-based compound that stores energy. Organic fuels include, but are not limited to, nucleic acids, carbohydrates (eg glucose), alcohols, fatty acids and other hydrocarbons, ketones, aldehydes, amino acids and proteins. The organic fuel may be a biological compound in the organism. Preferred fuels are alcohols including methanol, ethanol, butanol and isopropanol, and carbohydrates, especially glucose or polymers thereof. Preferred alcohols are ethanol and methanol.

  The invention also relates to a bioanode. A bioanode is an anode comprising an enzyme that catalyzes the oxidation of a fuel fluid. In one embodiment, “bioanode” refers to an anode comprising a redox enzyme that catalyzes the oxidation of an organic fuel. The anode provides an electron source for the electrical circuit or potential difference. In other embodiments, preferred bioanodes comprise a support membrane or structure, such as a carbon fiber cloth or a sheet of carbon felt, juxtaposed to a redox polymer membrane juxtaposed to an ion exchange polymer membrane. In one embodiment, the term “biocathode” comprises a redox enzyme such as laccase that catalyzes the reduction of oxygen.

As used herein, the terms “redox polymer”, “redox polymer film” or “redox polymer film” can accept or donate electrons from a compound, thereby oxidizing or reducing the compound, respectively. And free electrons that can be used to move to the electrical circuit. A preferred redox polymer is polymethylene green, as described in document 5 (Zou et al., 1996). Preferred compounds that are substrates for electrocatalysis by redox polymers include reduced adenine dinucleotides such as NADH, FADH ₂ and NADPH. Redox polymer films useful for biocathodes include poly (N-vinyl-imidazole) and its derivatives.

  As used herein, the term “support membrane” refers to a rigid or semi-rigid inert material that can conduct current and is used to support the polymer membrane of a biofuel cell electrode. The support membrane may comprise any conductive material, such as stainless steel, stainless steel mesh, carbon, carbon nanotubes, platinum or a semiconductor material. A preferred support membrane is a carbon felt sheet. The terms “carbon felt”, “carbon cloth” and “carbon cloth support membrane” are used interchangeably.

As used herein, the term “ion exchange polymer” or “ion exchange polymer membrane” means a polymer that can allow conduction of ions therethrough. Preferred ion exchange polymers are perfluorinated ion exchange polymers such as Nafion ^™ (DuPont, Wilmington, DE). The present invention also relates to perfluorinated ion exchange polymers having modifications containing quaternary ammonium ions at the sulfonic acid exchange sites. The modification results in a neutral pH in the ion exchange polymer micelles. In accordance with the present invention, one or more redox enzymes are incorporated or trapped in the micelles (or “micelle compartment”) of the salt extracted quaternary ammonium treated perfluorinated ion exchange polymer.

In one embodiment, the term “enzyme” or “redox enzyme” refers to a protein that acts as a catalyst in a chemical reaction. Preferred enzymes include “dehydrogenases” that catalyze the oxidation of fuel and the simultaneous reduction of “cofactors”. Preferred cofactors include adenine dinucleotides such as nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) and flavin adenine dinucleotide (FAD). Dehydrogenases include alcohol dehydrogenase, aldehyde dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, lactate dehydrogenase, glucose dehydrogenase and pyruvate dehydrogenase. Redox enzymes useful for biocathodes include O ₂ reductases such as laccase.

  As used herein, the terms “hydrocarbon” and “hydrocarbyl” mean an organic compound or group consisting solely of carbon and hydrogen. These components include alkyl, alkenyl, alkynyl and aryl components. These components also include alkyl, alkenyl, alkynyl and aryl components substituted with other aliphatic or cyclic hydrocarbon groups such as alkaryl, alkenaryl and alkynalyl. Unless otherwise specified, these components preferably have 1 to 20 carbon atoms.

  A “substituted hydrocarbyl” component as described herein is a hydrocarbyl component substituted with at least one atom other than carbon, where the carbon chain atoms are nitrogen, oxygen, silicon, phosphorus, boron, sulfur or halogen atoms. And a component substituted with a heteroatom such as These substituents include halogen, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protected hydroxy, keto, acyl, acyloxy, nitro, amino, amide, nitro, cyano, thiol, ketal, acetal, ester and ether. Include.

  The term “heteroatom” means atoms other than carbon and hydrogen.

  The term “heterocyclo” or “heterocyclic” as used herein alone or as part of another group preferably has at least one heteroatom in at least one ring and is preferably in each ring. Means 5 or 6 atoms, optionally substituted, means fully saturated or unsaturated, monocyclic or bicyclic, aromatic or non-aromatic group. The heterocyclo group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and / or 1 to 4 nitrogen atoms in the ring, and may be attached to the molecule via a carbon or heteroatom. Can bind to the rest. Illustrative heterocyclo includes heteroaromatic hydrocarbons such as furyl, thienyl, pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl or isoquinolinyl. Exemplary substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amide, amino, Nitro, cyano, thiol, ketal, acetal, ester and ether.

The following examples illustrate the invention.
Example 1
Immobilized enzyme
A casting solution for the production of a mixed casting membrane of Nafion ^™ and quaternary ammonium bromide was prepared as described in document 2, which is incorporated herein by reference. 1 mL of the mixed casting solution was placed in a weighing boat and dried. Previous tests have shown that all bromide ions introduced into the membrane were released from the membrane when the membrane was immersed in water [2]. Therefore, 7.0 mL of 18 MW water was added to the weighing boat and immersed overnight. The water was removed and the film was rinsed thoroughly with 18 MW water and dried. The salt extraction film was then resuspended in 1.0 mL of lower aliphatic alcohol.

Enzyme / Nafion ^™ casting solution and 0.03 g NAD ⁺ in an enzyme / TBAB ratio of 2: 1 (typically 1.0 μM enzyme 1200 μL / 600 μL) were vortexed in preparation for electrode coating. The solution was pipetted onto the electrode, immersed in a carbon felt electrode and dried.

Solution casting procedure The following quaternary ammonium bromides were used: ammonium bromide (Fisher), tetramethylammonium bromide (Aldrich), tetraethylammonium bromide (Fisher), tetrapropylammonium bromide (Aldrich), tetrabutylammonium. Umbromide (Eastman) and tetrapentylammonium bromide (Aldrich).

Nafion ^™ membranes incorporating quaternary ammonium bromide were formed by casting together quaternary ammonium bromide and 5 wt% Nafion ^™ suspension (Solution Technologies, Inc.). A mixed casting solution was prepared by adding quaternary ammonium bromide to a 5 wt% suspension. All mixed casting solutions were prepared such that the concentration of quaternary ammonium bromide was higher than the concentration of sulfonic acid sites in the Nafion ^™ suspension. After optimization, the most stable and reproducible membrane was determined to have a quaternary ammonium bromide concentration of 3 times the exchange site concentration.

  1 mL of the casting solution was placed in a weighing boat and dried. Previous tests have shown that all bromide ions introduced into the membrane were released from the membrane when the membrane was immersed in water. Therefore, 7.0 mL of 18 MW water was added to the weighing boat and immersed overnight. The water was removed and the film was rinsed thoroughly with 18 MW water and dried. The film was then resuspended in 1.0 mL methanol.

FIGS. 4-6 show fluorescence micrographs of enzymes immobilized on various modified perfluorosulfonic acid-PTFE copolymer membranes treated with a solution of NAD ⁺ and fuel fluid in pH 7.15 phosphate buffer. Fluorescence micrographs showing fluorescence show that the enzyme immobilized on the specific modified perfluorosulfonic acid-PTFE copolymer membrane is still catalytically active after immobilization. This is one way to determine whether a particular immobilization material will immobilize and stabilize the enzyme while maintaining the catalytic activity of the enzyme. The table below shows the prepared enzymes and membranes and whether the enzymes retained catalytic activity after immobilization. In the table, “Yes” indicates that the enzyme retains its catalytic activity after immobilization on the membrane, and “No” indicates that the enzyme retains its catalytic activity after immobilization on the membrane. Absent.

Example 2
Physical Battery Device The bioanode of the present invention was tested in a physical test cell consisting of a custom made “U” shaped cylindrical glass tube system of 2.6 cm diameter, 14.8 cm height and 7.6 cm length as shown in FIG. Approximately 50 mL of the solution was included on both sides of the Nafion ^™ membrane (Aldrich). The cathode side of the test cell contained a pH 7.15 phosphate buffer saturated with dissolved oxygen added by bubbling with an external source (AirGas). The cathode material is an ELAT electrode with 20% platinum on Vulcan XC-72 (E-Tek). The anode side of the test cell was filled with pH 7.15 buffer containing 1.0 mM NAD ⁺ and 1.0 mM fuel (ethanol or methanol). A bioanode according to the invention comprising an enzyme was used as the anode. Prior to data collection, the entire battery was allowed to equilibrate for 2-6 hours. All data was collected and analyzed for the test cells using a CH Instruments 810 potentiostat connected to a PC computer. Each type of biofuel cell was tested in triplicate and all uncertainties recorded correspond to one standard deviation.

Example 3
Bioanode for ethanol A bioanode for ethanol was designed as shown in FIG. These bioanodes have NAD ⁺ and alcohol dehydrogenase or a mixture of alcohol dehydrogenase and aldehyde dehydrogenase immobilized on a salt extracted tetrabutylammonium (“TBAB”) / Nafion ^™ membrane. The membrane is cast onto a poly (methylene green) modified carbon cloth electrode. These bioanodes can function for longer than 30 days, and this function is determined by more than 20% of the initial power generation of the fuel cell.

The bioanode using alcohol dehydrogenase immobilized on TBAB / Nafion ^™ membrane showed an open circuit potential difference of 0.60 to 0.62 V at 20 ° C. and buffer pH 7.15. The average maximum power density of ethanol-based biofuel cells is 1.16 ± 0.05 mW / cm ² . A typical power curve for an ethanol / O ₂ fuel cell is shown in FIG. The bioanode tested in FIG. 3 has 1.2 × 10 ⁻⁹ moles of alcohol dehydrogenase enzyme immobilized on a TBAB / Nafion ^™ membrane. The optimum pH for the enzyme is 8.6-9.0 [7], but poly (methylene green) electrocatalyst has the highest catalytic effectiveness for the oxidation of NADH at neutral pH [5], so pH 7.15 phosphate A salt buffer was selected. This power curve was taken after 2 days. Maximum power decreased 6.1% in the first 7 days.

A bioanode using a mixture of alcohol and aldehyde dehydrogenases immobilized on a TBAB / Nafion ^™ membrane was fabricated and tested. The bio-anode were immobilized alcohol dehydrogenase 1.2x10 ^-9 mol and aldehyde dehydrogenase 1.2x10 ^-9 mol to TBAB / Nafion ^TM membranes. The open circuit potential difference of an ethanol / O ₂ fuel cell using alcohol dehydrogenase and aldehyde dehydrogenase is 0.82V. The maximum power density of an ethanol-based fuel cell using an equimolar mixture of alcohol dehydrogenase and aldehyde dehydrogenase is 2.04 mW / cm ² .

In order to compare these alcohol / O ₂ biofuel cells with literature biofuel cells, methanol / O ₂ biofuel cells were manufactured and tested. Methanol Bio anode had TBAB / Nafion ^TM film immobilized alcohol dehydrogenase 1.2x10 ^-9 mol of formaldehyde dehydrogenase 1.2x10 ^-9 mol and formate dehydrogenase 1.2x10 ^-9 mol. Alcohol dehydrogenase oxidizes methanol to formaldehyde, which is then oxidized to formate by formaldehyde dehydrogenase. Finally, formate is oxidized to carbon dioxide by formate dehydrogenase, thereby producing 3 moles of NADH per mole of fuel reacted.

Current technology methanol-based biofuel cells have an open circuit potential difference of 0.64 to 0.71 V at 20 ° C. and buffer pH 7.15. The average maximum power density of a methanol-based biofuel cell is 1.55 ± 0.19 mW / cm ² . The current technology methanol / O ₂ biofuel cell developed by Whitesides and collaborators had an open circuit potential difference of 0.8 V and a maximum power generation of the cell of 0.67 mW / cm ² [8]. The methanol biofuel cell described in Document 1 does not have an immobilized enzyme, and therefore the life of the fuel cell is long and limited to a few days. The methanol biofuel cells of the present invention based on TBAB / Nafion ^™ membranes have been tested for more than 5 days without performance degradation.

While many other methanol biofuel cells have been shown in the literature [9-11], the highest open circuit potential difference reported is 0.3V. This is due to the combination of low redox mediator and low catalytic activity. Poly (methylene green) redox mediators are more stable than aqueous redox mediators or adsorbed redox mediators, reducing NADH oxidation overvoltage by 400 mV [5], and TBAB / Nafion ^TM membranes are ideal for enzyme immobilization Give a positive environment.

Example 4
Reagent and Electrode Production Methylene Green (Sigma), Sodium Nitrate (Fisher), Sodium Borate (Fisher), Nafion ^™ 1100 Suspension (Aldrich) and Nafion ^™ 117 (Aldrich) were purchased and used as received. The enzymes used were alcohol dehydrogenase (EC 1.1.1.1, initial activity 300-500 units / mg), aldehyde dehydrogenase (EC 1.2.1.5, initial activity 2-10 units / mg), formaldehyde dehydrogenase (EC 1.2.1.2, initial Activity 1-6 units / mg), and formate dehydrogenase (EC 1.2.1.46, initial activity 5-15 units / mg). Enzymes were purchased from Sigma (St. Louis, MO) and Roche Applied Science (Indianapolis, IN), stored at 0 ° C. and used as received. In addition, NAD ⁺ and NADH were purchased from Sigma and used as received.

  The bioanode consists of 1 cm x 1 cm square carbon felt (Alfa Aesar) electropolymerized with methylene green. In a solution containing 0.4 mM methylene green and 0.1 M sodium nitrate in 10 mM sodium tetraborate, CH Instruments 810 at -0.3 volts to 1.3 volts for 12 sweep segments at a scan rate of 0.05 V / sec. Poly (methylene green) thin films were formed by cyclic voltammetry using 620 potentiostat (Austin, TX). The electrode was rinsed and then allowed to dry overnight before further modification.

Examples 5-7
The same procedure used in Example 3, except that bioanode- designated enzymes and fuel fluids for various fuel fluids were used.

Example 8
Graphite worm methylene green (Sigma), sodium nitrate (Fisher) and sodium borate (Fisher) treated with polymethylene green were purchased and used as received. GAT expanded graphite worm, lab code: 3-96-16 (Superior Graphite Co.), non-waterproof carbon cloth (E-Tek) and carbon tape (Spi Supplies) were used as received.

  A methylene green-containing buffer was prepared in 18 MΩ water by adding 1 mM concentrations of methylene green, sodium nitrate and sodium borate. Prior to use, the solute was fully dissolved. Next, a desired amount of GAT expanded graphite worm was placed in a large container filled with an appropriate amount of methylene green buffer solution. The graphite and methylene green buffer solutions were mixed well for 30 minutes to ensure complete adsorption of methylene green on the graphite surface.

  The mixture of graphite and buffer solution was separated based on the low density of graphite in the solution. Next, excess buffer was flushed out of the graphite and the graphite was then washed with 18 MΩ water. After each wash, the solution was separated, excess was poured off, and additional washes were performed. This process was repeated until the wash solution became clear and did not appear to contain excess methylene green. The final wash was drained and the pretreated graphite material was completely dried before further use.

  The resulting material was similar to loose graphite powder and had to be processed into a usable electrode form. There was considerable flexibility in processing the final electrode to any desired size or shape. The parameter selected was to prepare a 2.5 cm x 2.5 cm electrode. This was done by filling a custom-made plexiglas mold of the desired dimensions with pretreated graphite material. The material was pressurized to varying pressures using a hydraulic jack. Generally 3000 psi was used.

  Any pressurization of worms below 3000 psi produced mechanically unstable electronic conductors. Generally, the electronic conductor is pressurized to 3000-4000 psi. This was sufficient mechanical stability to use without additional support to attach it to the carbon fiber. Furthermore, these pressurization conditions produced a semi-permeable electronic conductor in the liquid. The higher pressure resulted in an electron conductor that was too dense and the liquid was not easily absorbed by the electron conductor.

  The resulting electrode was rough and could be attached to a support material for further stability. The support material used was a carbon cloth. A layer of double-sided carbon tape was attached to one side of the carbon cloth, and a pretreated press graphite worm was placed thereon.

  At this point, the redox species was polymerized to improve the stability and lifetime of the redox species. The polymerization can be carried out by chemical or electrochemical means. In this case, the redox species was electrochemically polymerized by placing the constructed electrode into a solution containing 1 mM methylene green, 1 mM sodium nitrate and 1 mM sodium borate. The system was linked to a CH Instruments 810 potentiostat with a component electrode that served as the working electrode, a platinum counter electrode and an Ag / AgCl reference. For the 14 sweep segments, the potential difference was scanned from -0.3V to 1.3V and -0.3V. This resulted in a stable methylene green polymer on the electrode.

Example 9
Immobilized oxidases, particularly laccase enzymes, in biocathode quaternary ammonium-added Nafion ^™ films are similar to immobilized dehydrogenase enzymes. A series of quaternary ammonium salts is used to determine which salt provides optimal enzyme activity when the enzyme is immobilized. Enzymes generally use oxygen as an oxidant. Enzyme activity is measured electrochemically (cyclic voltammetry) in saturated oxygen environments in different buffers with different pH ranges, but due to the low electrochemical of peroxides in most electrode materials, Redox mediators are needed. Redox mediators tested are [Fe (bipyridine) ₃ ] ²⁺ , [Os (bipyridine) ₃ ] ²⁺ , [Ru (bipyridine) ₃ ] ²⁺ , and are physically stable and have reversible electrochemistry Including any other redox couple with a standard reduction potential difference of 0.8V to 2.0V. The redox mediator can be mixed cast with the membrane, salt extracted into the membrane prior to final casting, or extracted into the membrane after casting.

Example 10
Membrane electrode assembly (MEA)
The membrane electrode assembly (MEA) process began with the configuration of the bioanode. The bioanode can be composed of any of the carbon, graphite or metal electrode materials described above. The overall MEA process is unchanged.

When using a methylene green pretreated uncompressed graphite worm, electrodes were prepared as described above. The electrode was pressed to the desired specification under hydraulic pressure, electropolymerized with methylene green, coated with a modified Nafion ^™ / enzyme / coenzyme polymer, dried and then ready for use in MEA. The electrodes were generally coated with Nafion ^™ / enzyme / coenzyme polymer on both sides.

The MEA manufacturing process first included cleaning and laying Kapton (DuPont). Kapton prevents the MEA from sticking to the heated press and keeps the MEA clean. A platinum-added (single or double-sided) gas diffusion electrode (Etek) was placed on top of Kapton, on which Nafion ^™ 117, 112, or 111 cut approximately 2.5 times larger than the gas diffusion electrode was placed. A pre-manufactured bioanode was then placed on the Nafion ^™ membrane. In order to bond the assembly, the assembly was slightly moistened before placing another kapton on the bioanode.

  The MEA assembly was then placed between the hydraulic press heating elements. Enough pressure was applied so that the upper and lower platens of the press just began to contact. Heat was applied and the platen was heated to 120-135 ° C. When they reach that temperature, apply pressure to the desired standard, typically 3000-4000 psi. The platen can be preheated before placing the MEA assembly on the press. The procedure does not necessarily follow these steps exactly, and can be successfully performed with changes to the procedure.

  After 3-5 minutes under pressure, the MEA was removed and allowed to cool before separation from Kapton.

MEA can be formed without hot pressing. The MEA was similarly constructed except that a few drops of liquid Nafion ^TM or salt-modified Nafion ^TM were added both between the anode and Nafion ^TM 117, 112 or 111, and between the cathode and Nafion ^TM 117, 112 or 111. . The assembly was then pressed without heating until the liquid Nafion ^™ was dry.

  In addition, the anode can be pre-manufactured as follows. When a redox polymer was required, the desired electrode material was electropolymerized or chemically polymerized into a redox polymer. At this time, electrodes were placed on the MEA assembly without placing the enzyme and hot pressed as described above. After pressing and cooling, the enzyme was cast to the anode side of the finished MEA.

Nine biofuel cells MEA were manufactured, 7 of which worked properly. The open circuit potential difference produced by the biofuel cell MEA was 0.36 to 0.599 V and the power density was 0.15 to 1.49 mW / cm ² .

Example 11
An example of a coenzyme that can be oxidized at a PQQ-dependent enzyme non-denaturing electrode is pyrroloquinoline quinone (PQQ). PQQ was found as a redox cofactor in bacterial alcohol dehydrogenase. PQQ contains an o-quinone component that exhibits effective pH-dependent and reversible electron transfer between its oxidized and reduced forms at the unmodified carbon-based electrode. The use of a PQQ-dependent dehydrogenase enzyme makes it possible to remove the electrocatalyst used in biofuel cells and sensors. This simplifies electrode construction, reduces cost, and lower power loss due to slow mass transfer due to the higher flux of PQQ through the membrane due to the smaller molecular size.

Example 12
Glassy carbon electrodes were modified with methylene green polymerized by enzyme sensor chemical or electrochemical means. Using the procedure described in Example 1, alcohol dehydrogenase was immobilized on a tetrabutylammonium bromide modified Nafion ^™ membrane, and the immobilized enzyme was layered on a modified glassy carbon electrode and dried. The modified glassy carbon electrode was dried and then equilibrated in 1.5 mL of pH 7.15 phosphate buffer. After equilibration, amperometric detection was performed with a potential difference of −0.4V. After establishing the baseline reading, 1 mL of an 8 mM ethanol solution in phosphate buffer was injected into the solution. FIG. 10 shows a current versus time graph for an amperometric sensor.

Description of Related Literatures References cited throughout this specification are listed here by their number and are hereby incorporated by reference. The discussion of the documents listed here is only a summary of the claims made by the authors of the documents and does not admit that any document constitutes prior art. Applicant reserves the right to claim the accuracy and validity of the cited document:
1． GTR Palmore; GM Whitesides, "Microbial and Enzymatic Biofuel Cells", ACS Symposium Series 566 (1994) 271-290.
2． M. Schrenk; R. Villigram; N. Torrence; S. Brancato; SD Minteer, "Effect of Mixture Casting Nafion ^TM with Quaternary Ammonium Bromide Salts on the Ion-Exchange Capacity and Mass Transport in the Membranes", Journal of Membrane Science 205 (2002) 3-10.
3． TJ Thomas; KE Ponnusamy; NM Chang; K. Galmore; SD Minteer, "Effects of Annealing on Mixture-Cast Membranes of Nafion ^TM and Quaternary Ammonium Bromide Salts", Journal of Membrane Science, Vol. 213, (2003) 55-56 .
Four. RA Jenkins, “Study of the Electrochemical Oxidation of Reduced Nicotinamide Adenine Dinucleotide”, Analytical Chemistry 47 (1975) 1337-1338.
Five. H. Fang; H. Chen; H. Ju; Y. Wang, “The Electrochemical Polymerization of Methylene Green and its Electrocatalysis for the Oxidation of NADH”, Analytica Chimica Acta 329 (1996) 41-48.
6． DW Green; HW Sun; BV Plapp, “Inversion of the Substrate Specificity of Yeast Alcohol Dehydrogenase”, Journal of Biological Chemistry 268 (1993) 7792-7798.
7． Worthington, V. "Worthington Enzyme Manual" (1988) 16.
8． GTR Palmore; H. Bertschy; SH Bergens; GM Whitesides, "A Methanol / Dioxygen Biofuel Cell that Uses NAD ⁺ -Dependent Dehydrogenases as Catalysts: Application of an Electro-Enzymic Method to Regenerate Nicotinamide Adenine Dinucleotide at Low Overpotentials", Journal of Electroanalytical Chemistry 443 (1998) 155-161.
9． EV Plotkin, IJ Higgins; HAO Hill, "Methanol Dehydrogenase Bioelectrochemical Cell and Alcohol Detector", Biotechnology Letters 3 (1981) 187-192.
Ten. WJ Aston; IJ Higgins; APF Turner, "Bioelectrochemical Fuel-Cell and Sensor Based on a Quinoprotein, Alcohol-Dehydrogenase", Enzyme and Microbial Technology 5 (1983) 383-388.
11． PL Yue; K. Lowther, “Enzymatic Oxidation of C1 Compounds in a Biochemical Fuel Cell”, Chemical Engineering Journal. 33B (1986) 69-77.

  In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results are obtained.

  Since various modifications can be made to the above method without departing from the scope of the present invention, all content contained in the foregoing description and shown in the accompanying drawings is illustrative. It is understood that this is not a limitation.

1 is a schematic diagram (not a fixed ratio) of a biofuel cell. Oxidation of ethanol to aldehyde is catalyzed by NAD ⁺ dependent alcohol dehydrogenase (ADH). NADH is electrolyzed at the poly (methylene green) modified electrode. Figure _{2 is} a typical power curve for an ethanol / O2 biofuel cell at room temperature with an open circuit voltage of 0.60V. FIG. 5 is a series of fluorescence micrographs of NADH formation in alcohol dehydrogenase immobilized on a modified Nafion ^™ membrane treated with NAD ⁺ and ethanol in pH 7.15 phosphate buffer. In the figure, (a) is a native Nafion ^™ membrane, (b) is a tetramethylammonium bromide / Nafion ^™ membrane, (c) is a tetraethylammonium bromide / Nafion ^™ membrane, and (d) is a tetrapropyl ammonium. Ammonium bromide / Nafion ^™ membrane, (e) is a tetrabutylammonium bromide / Nafion ^™ membrane, and (f) is a tetrapentylammonium bromide / Nafion ^™ membrane. Fluorescence micrograph of annealed alcohol dehydrogenase immobilized on tetrabutylammonium bromide / Nafion ^™ membrane treated with NAD ⁺ and ethanol solution in pH 7.15 phosphate buffer. Fluorescence micrograph of aldehyde dehydrogenase immobilized on tetrapentylammonium bromide / Nafion ^™ membrane treated with NAD ⁺ and acetaldehyde solution in pH 7.15 phosphate buffer. 1 is a schematic diagram of a biocathode using laccase immobilized on a Nafion ^™ membrane. FIG. Figure 2 is a cyclic voltammogram comparing fuels for a tetrabutylammonium bromide / Nafion ^™ membrane. It is a cyclic voltammogram of pyrroloquinoline quinone (PQQ) in a bare glassy carbon electrode. It is a graph which shows the electric current versus time curve about an ethanol sensor. 1 is a schematic view of a membrane electrode assembly (MEA) for a biofuel cell. FIG. ₂ is a typical power curve for an ethanol / O ₂ membrane electrode assembly (MEA) biofuel cell operating at room temperature providing an open circuit voltage of 0.599V.

Claims (14)

(A) electronic conductors,
(B) at least one enzyme capable of reacting with the oxidized form of the electron mediator and the fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator;
(C) can be of immobilizing and stabilizing the enzyme, an enzyme immobilization material comprising a micellar or inverted micellar structure, the fuel fluid and the electron mediator permeability der is, enzymes that are stabilized, at least 30 An enzyme immobilization material that retains at least 75% of the initial enzyme activity over a period of time , and (d) an electrocatalyst in proximity to the electron conductor, wherein the oxidized form of the electrocatalyst reacts with the reduced form of the electron mediator; A bioanode having an electrocatalyst capable of producing an oxidized form of an electron mediator and a reduced form of an electrocatalyst, wherein the reduced form of the electrocatalyst can emit electrons to an electron conductor. (A) electronic conductors,
(B) at least one enzyme capable of reacting with the oxidized form of the electron mediator and the fuel fluid to produce an oxidized form of the fuel fluid and a reduced form of the electron mediator;
(C) has the micellar or inverted micellar structure, an enzyme immobilization material comprising the electron mediator, able of immobilizing and stabilizing the enzyme, the fuel fluid permeable der is, stabilized The enzyme retains at least 75% of the initial enzyme activity for at least 30 days , and (d) an electrocatalyst proximate to the electron conductor, wherein the oxidized form of the electrocatalyst is the reduced form of the electron mediator A bioanode having an electrocatalyst that can be reacted with to produce an oxidized form of an electron mediator and a reduced form of an electrocatalyst, wherein the reduced form of the electrocatalyst can release electrons to an electron conductor. (A) electronic conductors,
(B) It can react with the oxidized form of the electron mediator and the fuel fluid to produce the oxidized form of the fuel fluid and the reduced form of the electron mediator, and the reduced form of the electron mediator can emit electrons to the electron conductor. at least one enzyme, and (c) can be of immobilizing and stabilizing the enzyme, an enzyme immobilization material comprising a micellar or inverted micellar structure, Ri fuel fluid and the electron mediator permeability der, stabilized The enzyme-immobilized material that retains at least 75% of the initial enzyme activity for at least 30 days
Bio-anode comprising a. (A) electronic conductors,
(B) It can react with the oxidized form of the electron mediator and the fuel fluid to produce the oxidized form of the fuel fluid and the reduced form of the electron mediator, and the reduced form of the electron mediator can emit electrons to the electron conductor. At least one enzyme; and (c) an enzyme immobilization material having a micelle structure or reverse micelle structure and comprising an electron mediator, capable of immobilizing and stabilizing the enzyme, and being permeable to fuel fluid A bioanode having an enzyme immobilization material wherein the stabilized enzyme retains at least 75% of the initial enzyme activity for at least 30 days .   The bioanode according to any one of claims 1 to 4, wherein the enzyme-immobilized substance comprises a modified perfluorosulfonic acid-polytetrafluoroethylene (PTFE) copolymer. A biofuel cell for generating electricity,
Fuel fluid;
Electronic mediators;
A biofuel cell comprising a cathode capable of reducing water to produce oxidant in the presence of electrons; and a bioanode according to claim 1 . A biofuel cell for generating electricity,
  Fuel fluid;
  Electronic mediators;
  A cathode capable of reducing the oxidant in the presence of electrons to produce water; and
  The bioanode according to claim 3.
A biofuel cell comprising: A biofuel cell for generating electricity,
Fuel fluid;
A cathode capable of reducing water to produce oxidant in the presence of electrons; and a biofuel cell comprising a bioanode according to claim 2 . A biofuel cell for generating electricity,
  Fuel fluid;
  A cathode capable of reducing the oxidant in the presence of electrons to produce water; and
  The bioanode according to claim 4.
A biofuel cell comprising: Electron conductor is carbon cloth, carbon paper, carbon screen printing electrode, carbon black, carbon powder, carbon fiber, single-walled carbon nanotube, double-walled carbon nanotube, multi-walled carbon nanotube, carbon nanotube array, diamond-coated conductor, glassy carbon, Consisting of mesoporous carbon, graphite, uncompressed graphite worm, delaminated flake graphite, high performance graphite, highly ordered pyrolytic graphite, pyrolytic graphite or polycrystalline graphite,
Enzyme immobilization material is ammonium based cation, quaternary ammonium cation, alkyltrimethylammonium cation, organic cation, phosphonium cation, triphenylphosphonium, pyridinium cation, imidazolium cation, hexadecylpyridinium, It consists of ethidium, viologen, methyl viologen, benzyl viologen, bis (triphenylphosphine) iminium, metal complex, bipyridyl metal complex, metal complex based on phenanthroline, [Ru (bipyridine) ₃ ] ²⁺ or [Fe (phenanthroline) ₃ ] ³⁺ Modified with a larger hydrophobic cation than NH ₄ ⁺
The enzyme comprises alcohol dehydrogenase, aldehyde dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, glucose dehydrogenase, glucose oxidase, lactate dehydrogenase, lactose dehydrogenase or pyruvate dehydrogenase;
Electrocatalysts for electron mediators include methylene green, methylene blue, luminol, nitro-fluorenone derivatives, azine, osmium phenanthrolinedione, catechol-pendant terpyridine, toluene blue, cresyl blue, nile blue, neutral red, phenazine derivatives, thionine, azure A , Azure B, Toluidine Blue O, Acetophenone, Metallophthalocyanine, Nile Blue A, Modified transition metal ligand, 1,10-phenanthroline-5,6-dione, 1,10-phenanthroline-5,6-diol, [Re (phen - _{dione) (CO) 3 Cl],} [Re ( phen - dione) _3] (PF _{6) 2,} poly (metallophthalocyanine), poly (thionine), quinones, diimines, diaminobenzenes, diaminopyridines, phenothiazine, phenoxazine , Toluidine blue, brilliant cresyl blue, 3,4-dihydroxybenzaldehyde, poly (acrylic acid), poly (azur I), poly (nile blue A), poly (methylene green), poly (methylene blue), polyaniline, polypyridine, Polypyrrole, polythiophene, poly (thieno [3,4-b] thiophene), poly (3-hexylthiophene), poly (3,4-ethylenedioxypyrrole), poly (isothianaphthene), poly (3,4- Ethylenedioxythiophene), poly (difluoroacetylene), poly (4-dicyanomethylene-4H-cyclopenta [2,1-b; 3,4-b ′] dithiophene), poly (3- (4-fluorophenyl) thiophene ) Or poly (neutral red)
The bioanode according to claim 1 or 2, or claim 6 or 8 , wherein the electron mediator comprises nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) or nicotinamide adenine dinucleotide phosphate (NADP). The biofuel cell as described. Electron conductor is carbon cloth, carbon paper, carbon screen printing electrode, carbon black, carbon powder, carbon fiber, single-walled carbon nanotube, double-walled carbon nanotube, multi-walled carbon nanotube, carbon nanotube array, diamond-coated conductor, glassy carbon, Made of mesoporous carbon, graphite, uncompressed graphite worm, delaminated flake graphite, high performance graphite, highly ordered pyrolytic graphite, pyrolytic graphite or polycrystalline graphite,
Enzyme immobilization material is ammonium based cation, quaternary ammonium cation, alkyltrimethylammonium cation, organic cation, phosphonium cation, triphenylphosphonium, pyridinium cation, imidazolium cation, hexadecylpyridinium, It consists of ethidium, viologen, methyl viologen, benzyl viologen, bis (triphenylphosphine) iminium, metal complex, bipyridyl metal complex, metal complex based on phenanthroline, [Ru (bipyridine) ₃ ] ²⁺ or [Fe (phenanthroline) ₃ ] ³⁺ Modified with a larger hydrophobic cation than NH ₄ ⁺
The enzyme comprises alcohol dehydrogenase, aldehyde dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, glucose dehydrogenase, glucose oxidase, lactate dehydrogenase, lactose dehydrogenase or pyruvate dehydrogenase;
The bioanode according to claim 3 or 4, or the biofuel cell according to claim 7 or 9 , wherein the electron mediator comprises pyrroloquinoline quinone, phenazine methosulfate, dichlorophenolindophenol, short chain ubiquinone or potassium ferricyanide. (A) oxidizing the fuel fluid at the bioanode and reducing the oxidant at the cathode;
(B) reducing the oxidized form of the electron mediator during oxidation of the fuel fluid at the bioanode;
(C) reducing the electrocatalyst;
11. A method of generating electricity using a biofuel cell according to claim 6, 8 or 10 , comprising (d) oxidizing an electrocatalyst in an electronic conductor. (A) oxidizing the fuel fluid at the bioanode and reducing the oxidant at the cathode;
(B) reducing the oxidized form of the electron mediator during oxidation of the fuel fluid at the bioanode;
(C) comprising oxidizing the electron mediator in the electronic conductor, a method of generating electricity using the biofuel cell according to claim 7, 9 or 11. The bioanode according to any one of claims 1 to 4, wherein the enzyme is captured by an enzyme-immobilized substance.

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